This invention relates to confocal microscopy in general, and particularly relates to the design of pinhole and slit detection aperture assemblies used in confocal microscopy.
Conventional wide field light microscopes create images whose effective depth of field at high coder is 2-3 xcexcm. Since the resolving power of optical microscopy is about 0.2 xcexcm, superimposition of detail within this plane of focus obscures structural detail that would otherwise be resolved. In addition, for specimens thicker than this depth of field, light from out-of-focus planes creates diffuse halos around objects under study. These halos are especially prominent in fluorescence microscopy. Confocal microscopy eliminates these undesirable artifacts by generating thin sub micron optical slices through thick specimens (M. Minsky, U.S. Pat. No. 3,013,467; M. Petran et al., J Opt. Soc. Am. 58, 661-664 (1968); J. White et al., J Cell Biol. 105, 41 (1987); T. Wilson, Confocal Microscopy (Academic Press, London 1990)). Confocal sections minimize superimposition of detail and exclude light from out-of-focus planes. As a consequence, images or remarkable detail and resolution are generated. Recently, ultraviolet-visible laser scanning confocal microscopes have become commercially available that expand the range of confocal applications to include UV-excited fluorophores. Increasingly, confocal microscopy has become an essential analytical tool in biology, medicine, materials science and microelectronics.
A confocal microscope scans a focused spot of light across the specimen (FIG. 1). Spot diameter is diffraction limited, or about 0.2 xcexcm for a high numerical aperture objective lens. Light fluoresced or reflected from the specimen is separated from the illuminating beam of light by a mirror or dichroic reflector and is focused by the objective lens onto a pinhole aperture. Light from above and below the focal plane misses the pinhole opening and strikes the wall of the aperture instead (FIG. 1). Thus, only light from a narrow in-focus plane passes through the pinhole to strike a photodetector beyond. In this way, the photodetector xe2x80x9cseesxe2x80x9d light from only a very narrow plane of focus.
Two dimensional images are generated as the illuminating spot of light moves across the specimen. Such scanning is achieved using vibrating mirrors, acousto-optical modulators, or a rotating disk containing multiple pinholes in a spiral arrangement (Nipkow disk). Reflected and fluoresced light passes back through the scan generator, a process that xe2x80x9cdescansxe2x80x9d the returning light so that it can be focused on a detection pinhole and transmitted to a photomultiplier. In laser scanning confocal microscopy, the instantaneous response of the is photomultiplier is then displayed on the synchronously scanned phosphor screen of a cathode ray tube monitor to recreate the image. Using a Nipkow disk in what is called tandem-scanning confocal microscopy, confocal images are viewed directly and recorded by photographic film (Petran et al., 1968). In certain configurations, a slit detection aperture replaces the pinhole detection aperture with only modest loss of Z-axis resolving power. In laser scanning confocal microscopy, images are typically stored in commuter memory for later analysis (white et al., 1987).
Confocal microscopes produce optical slices of defined thickness through thick specimens. For a high numerical aperture lens, thickness of the confocal sections can reach a theoretical limit of about 0.5 xcexcm. The thickness or confocal sections decreases as the detector pinhole is made smaller. Since not all applications require the thinnest possible confocal section, sensitivity can be increased by opening the pinhole aperture. Doubling the diameter of the pinhole quadruples sensitivity, but only about doubles the thickness of the optical slice. For this reason, most laser scanning confocal microscopes are equipped with variable pinhole apertures. For light sensitive specimens, a larger pinhole setting may be desirable so that laser power can be attenuated to an acceptable level. Conversely, pinhole diameter can be decreased to reduce slice thickness and increase resolution in the z-axis. However, below a minimum pinhole size, confocal slice thickness no longer decreases as pinhole diameter decreases, although image intensity continues to decline. Thus, overly small pinhole diameters should be avoided, especially with light sensitive specimens.
Although confocal microscopes achieve a Z-axis resolution approaching 0.5 xcexcm, this is not the diffraction limit of optical resolution for a high resolution microscope system, which is about 0.2 xcexcm An object of the present invention is to modify the detection aperture assembly to improve Z-axis resolution beyond that achievable by current technology.